Method for determining whether or not all of pythiums contained in test sample are non-phytopathogenic

ABSTRACT

The present invention provides a method for determining whether or not all of pythiums contained in a test sample are non-phytopathogenic. The method comprises: (a) putting the test sample on a front surface of a film comprising a through-hole having a cross-sectional area of not less than 0.785 square micrometers and not more than 7.065 square micrometers; (b) leaving the test sample at rest after the step (a); (c) observing a back surface of the film after the step (b); and (d) determining that all of the pythiums contained in the test sample are non-phytopathogenic, if pseudohyphae are not found on the back surface of the film in the step (c).

BACKGROUND 1. Technical Field

The present invention relates to a method for determining whether or notall of pythiums contained in a test sample are non-phytopathogenic.

2. Description of the Related Art

Patent Literature 1 discloses a method for counting the number of moldcells. FIG. 14 shows a cross-sectional view of a microporous membranesupporting material which is used for the method disclosed in PatentLiterature 1. The method for counting the number of mold cells disclosedin Patent Literature 1 provides a method for counting the number of moldcells in a specimen by the culture for a short time and capable ofaccurately counting the cell number. In the method for counting thenumber of mold cells disclosed in Patent Literature 1, the extendedmultiple pseudomycelia of a mold cell 13 cultured by a liquid culture ora mold cell 13 cultured on a microporous membrane 1 of a microporousmembrane supporting material 4 are photographed 5 and the shape, areaand luminous intensity are recognized and analyzed by an image analyticmeans 10. The number of the mold cells 13 can be counted by the culturefor a short time. The microporous membrane 1 is interposed between apressing ring 2 and a base 3.

Non-patent Literature 1 discloses that pseudohyphae of Phytophthorasojae, which is one kind of phytopathogenic pythiums, penetrate a PETfilm having pores each having a dimeter of 3 micrometers.

CITATION LIST Patent Literature

Patent Literature 1: Japanese Patent Application laid-open PublicationNo. 2005-287337A

Non-Patent Literature

Non-patent Literature 1: Paul F. Morris. et. al. “Chemotropic andContact Responses of Phytophthora sojae Hyphae to Soybean Isoflavonoidsand Artificial Substrates”, Plant Physiol. (1998) 117: 1171-1178

SUMMARY

An object of the present invention is to provide a method fordetermining whether or not all of pythiums contained in a test sampleare non-phytopathogenic.

The present invention provides a method for determining whether or notall of pythiums contained in a test sample are non-phytopathogenic, themethod comprising:

-   -   (a) putting the test sample on a front surface of a film        comprising a through-hole having a cross-sectional area of not        less than 0.785 square micrometers and not more than 7.065        square micrometers;    -   (b) leaving the test sample at rest after the step (a);    -   (c) observing a back surface of the film after the step (b); and    -   (d) determining that all of the pythiums contained in the test        sample are non-phytopathogenic, if pseudohyphae are not found on        the back surface of the film in the step (c).

The present invention provides a method for determining whether or notall of pythiums contained in a test sample are non-phytopathogenic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross-sectional view of a container.

FIG. 2 shows a cross-sectional view of a film.

FIG. 3 shows a cross-sectional view of the container to which a testsample has been supplied.

FIG. 4 shows a cross-sectional view of the film having a front surfaceon which a phytopathogenic pythium has been put.

FIG. 5 is a cross-sectional view showing how the phytopathogenic pythiumpenetrates the film.

FIG. 6 shows a cross-sectional view of one example of a method foraccelerating the incubation of the pythium.

FIG. 7 shows a cross-sectional view, subsequently to FIG. 6, of oneexample of a method for accelerating the incubation of the pythium.

FIG. 8 is a cross-sectional view showing how to observe the pythium fromthe back surface of the film.

FIG. 9 is a cross-sectional view showing how to observe the pythium fromthe back surface of the film.

FIG. 10 is a microscope photograph of the back surface of the film inthe inventive example 1A.

FIG. 11 is a microscope photograph of the back surface of the film inthe inventive example 1 B.

FIG. 12 is a microscope photograph of the back surface of the film inthe comparative example 1C.

FIG. 13 is a microscope photograph of the back surface of the film inthe comparative example 1D.

FIG. 14 shows a cross-sectional view of the microporous membranesupporting material used for the method for counting the number of moldcells disclosed in Patent Literature 1.

DETAILED DESCRIPTION OF THE EMBODIMENT

First, pythiums will be described. Pythiums are roughly divided into aphytopathogenic pythium and a non-phytopathogenic pythium. An example ofthe phytopathogenic pythium is Pythium helicoides, Pythium myliotaerum,or Pythium aphanidermatum. These phytopathogenic pythiums cause pythiumred blight and a root rot disease. First, these phytopathogenic pythiumsinfect a root of a plant. Then, these phytopathogenic pythiums cause theroot of the plant to rot. Finally, these phytopathogenic Pythiums killthe plant. An example of the non-phytopathogenic pythium is Pythiumdissotocum, Pythium catenulatum, Pythium torulosum, or Pythium inflatum.Pythium dissotocum may be classified as a weak-phytopathogenic pythium.In the instant specification, the weak-phytopathogenic pythium isclassified as a non-phytopathogenic pythium. In other words, the word“non-phytopathogenic pythium” includes a weak-phytopathogenic pythium.The word “phytopathogenic pythium” does not include aweak-phytopathogenic pythium.

The term “phytopathogenic” means to have pathogenicity to plants. Theterm “non-phytopathogenic” means not to have pathogenicity to plants.Even if a pythium has pathogenicity, however, if the pythium has nopathogenicity to plants, the pythium is non-phytopathogenic. In otherwords, if a pythium does not have adverse effects on plants, the pythiumis non-phytopathogenic. The prefix “non-” included in the term“non-phytopathogenic” does not modify “phyto”. The prefix “non-”modifies “pathogenic”.

Hereinafter, the embodiment of the present invention will be describedin more detail with reference to the drawings.

(Step (a))

In the step (a), a test sample is put on a front surface of a filmcomprising a through-hole having a cross-sectional area of not less than0.785 square micrometers and not more than 7.065 square micrometers.

In particular, as shown in FIG. 1, a container 100 is prepared. It isdesirable that the container 100 comprises a flange 102 at the upper endthereof. The bottom surface of the container 100 is formed of a film104. An example of the material of the film 104 is organic resin such aspolyethylene terephthalate.

FIG. 2 shows a cross-sectional view of the film 104. The film 104 has afront surface 104 a, a back surface 104 b, and a through-hole 104 c. Oneof the characteristics of the present invention is a cross-sectionalarea of the through-hole 104 c.

The through-hole 104 c has a cross-sectional area of not less than 0.785square micrometers and not more than 7.065 square micrometers. Inparticular, it is desirable that the through-hole 104 c has a shape of acylinder having a diameter of not less than 1 micrometer and not morethan 3 micrometers. The importance of these cross-sectional area anddiameter will be described later.

As shown in FIG. 3, a test sample 200 is supplied to the inside of thiscontainer 100. In this way, the test sample 200 is put on the frontsurface 104 a of the film 104. In the present embodiment, the testsample 200 contains at least one kind of pythiums. Therefore, as shownin FIG. 4, a pythium 202 is disposed on the front surface 104 a of thefilm 104.

The test sample 200 is solid, liquid, or gaseous. It is desirable thatthe test sample 200 is solid or liquid. An example of the solid testsample 200 is soil or a crushed plant. Another example is anagricultural material such as vermiculite, rock wool or urethane. Anexample of the liquid test sample 200 is agricultural water, a solutionused for hydroponic culture, a liquid used to wash a plant, a liquidextracted from a plant, a liquid used to wash an agricultural material,or a liquid used to wash clothing or shoes of a worker.

(Step (b))

In the step (b), the test sample 200 is left at rest for a certain timeafter the step (a). The importance of the cross-sectional area or thediameter of the through-hole 104 c will be described below.

In the step (b), various pythiums contained in the test sample 200 aregrown. When the through-hole 104 c has a cross-sectional area of notless than 0.785 square micrometers and not more than 7.065 squaremicrometers, as shown in FIG. 5, the phytopathogenic pythium 202 growsup so as to penetrate the through-hole 104 c. This is demonstrated inthe inventive examples and the comparative examples which will bedescribed later. As a result, pseudohyphae of the phytopathogenicpythium 202 appear on the back surface 104 b of the film 104. On theother hand, within this range of the cross-sectional area, thenon-phytopathogenic pythium does not penetrate the through-hole 104 c.For this reason, the non-phytopathogenic pythium does not appear on theback surface 104 b of the film 104. In this way, the phytopathogenicpythium 202 appears on the back surface 104 b selectively. In otherwords, the phytopathogenic pythium 202 appears outside of the container100 selectively.

When the through-hole 104 c has a cross-sectional area less than 0.785square micrometers, neither the non-phytopathogenic pythium nor thephytopathogenic pythium penetrates the through-hole 104 c. On the otherhand, when the through-hole 104 c has a cross-sectional area more than7.065 square micrometers, not only the phytopathogenic pythium but alsothe non-phytopathogenic pythium penetrate the through-hole 104 c. Forthis reason, the selectivity is lost when the through-hole 104 c has across-sectional area less than 0.785 square micrometers or more than7.065 square micrometers.

The thickness of the film 104 is not limited, as far as thephytopathogenic pythium 202 appears outside of the container 100selectively. The film 104 may have a thickness of not less than 10micrometers and not more than 100 micrometers. It is desirable that thefilm 104 has plural through-holes 104 c, as shown in FIG. 3-FIG. 5.

A culture medium may be supplied to the test sample 200 to acceleratethe incubation of the pythiums. In particular, a culture medium may besupplied to the inside of the container 100 containing the test sample200. It is desirable that the culture medium is liquid. The culturemedium may be supplied in the step (b). Alternatively, the culturemedium may be supplied prior to the step (b). In other words, theculture medium may be supplied in the step (a). The culture medium maybe supplied to the inside of the container 100 prior to the step (a).

FIG. 6 shows another method for accelerating the incubation of thepythiums. As shown in FIG. 6, it is desirable that the back surface 104b of the film 104 is in contact with a liquid culture medium 302. First,a second container 300 having the liquid culture medium 302 therein isprepared. Hereinafter, the container 100 is referred to as “firstcontainer 100” to distinguish it from the second container 300. Thefirst container 100 is stacked on the second container 300 in such amanner that the lower surface of the flange 102 is in contact with theupper end of the second container 300. In other words, the firstcontainer 100 is supported by the upper end of the second container 300.In this way, the liquid culture medium 302 is sandwiched between theback surface 104 b of the film 104 and the bottom surface of the secondcontainer 300.

Alternatively, after the first container 100 is stacked on the secondcontainer 300, the liquid culture medium 302 may be supplied between theback surface 104 b of the film 104 and the bottom surface of the secondcontainer 300.

Since the liquid culture medium 302 is in contact with the back surface104 b of the film 104, the liquid culture medium 302 is soaked up by acapillary phenomenon through the through-hole 104 c. In place of theliquid culture medium 302, a viscous solid culture medium may also beused. In this case, when the first container 100 is stacked on thesecond container 300, the viscous solid culture medium is transformed soas to penetrate the through-hole 104 c. In this way, the culture medium302 reaches the inside of the container 100. By the culture medium 302which has reached the inside of the container 100, the incubation of thepythiums is accelerated. As shown in FIG. 6, both of a solid culturemedium 304 and the liquid culture medium 302 may be used. In this case,the liquid culture medium 302 is sandwiched between the solid culturemedium 304 and the film 104.

(Step (c))

In the step (c), the back surface 104 b of the film 104 is observedafter the step (b). It is desirable that the back surface 104 b isobserved using an optical microscope.

The phytopathogenic pythium 202 appears on the back surface 104 b of thefilm 104, as described in the step (b). On the other hand, thenon-phytopathogenic pythium does not appear on the back surface 104 b ofthe film 104. In this way, in the present invention, the phytopathogenicpythium 202 appears on the back surface 104 b of the film 104selectively.

In other words, the phytopathogenic pythium 202 penetrates thethrough-hole 104 c, whereas the non-phytopathogenic pythium does notpenetrate the through-hole 104 c. For this reason, thenon-phytopathogenic pythium does not appear on the back surface 104 b ofthe film 104. In this way, the phytopathogenic pythium 202 appears onthe back surface 104 b selectively. In other words, the phytopathogenicpythium 202 appears outside of the container 100 selectively.

In the step (c), it is observed whether or not the phytopathogenicpythium 202 appears on the back surface 104 b of the film 104.

In particular, whether or not the phytopathogenic pythium 202 appears onthe back surface 104 b of the film 104 is observed as below.

First, the test sample 200 is turned into a gel. In more detail, anagarose aqueous solution is supplied to the first container 100. Then,the agarose aqueous solution containing the test sample 200 is stirred.Finally, the test sample 200 is left at rest at room temperature. Inthis way, the test sample is turned into a gel.

Then, the first container 100 is drawn up from the second container 300.Prior to the gelation, the first container 100 may be drawn up from thesecond container 300.

The liquid culture medium 302 and the solid culture medium 304 areremoved from the second container 300. Then, a fluorescent agent havingoomycete combining ability is added to the inside of the secondcontainer 300. Hereinafter, such a fluorescent agent is referred to as“oomycete fluorescent agent”. The reference number of the oomycetefluorescent agent is 402. Then, as shown in FIG. 7, the first container100 is stacked on the second container 300 having the oomycetefluorescent agent 402 therein. Alternatively, the oomycete fluorescentagent 402 may be supplied between the back surface 104 b of the film 104and the bottom surface of the second container 300 after the firstcontainer 100 is stacked on the second container 300.

A part of the phytopathogenic pythium 202 which has appeared on the backsurface 104 b of the film 104 is dyed with the oomycete fluorescentagent 402. Since the test sample 200 has been turned into a gel, theoomycete fluorescent agent 402 does not spread into the first container100. For this reason, the non-phytopathogenic pythium contained in thefirst container 100 is not dyed with the oomycete fluorescent agent 402.

As shown in FIG. 8, the thus-dyed phytopathogenic pythium 202 isobserved using a microscope 600 located under the back surface 104 b ofthe film 104, while the film 104 is irradiated with light using a lightsource 500 located over the front surface 104 a of the film 104.

In place of the oomycete fluorescent agent 402, a fluorescent agenthaving pythium combining ability may also be used. In this case, a part202 a of the phytopathogenic pythium 202 which has appeared on the backsurface 104 b of the film 104 is dyed with the fluorescent agent havingpythium combining ability. As shown in FIG. 9, the phytopathogenicpythium 202 dyed with the fluorescent agent having pythium combiningability is observed using the microscope 600 located under the backsurface 104 b of the film 104.

(Step (d))

In the step (d), it is determined that all of the pythiums contained inthe test sample 200 are non-phytopathogenic, if pseudohyphae of thepythiums are not found on the back surface 104 b of the film 104 in thestep (c). Needless to say, it is determined that all of the pythiumscontained in the test solution 200 are not necessarilynon-phytopathogenic, if the pythiums are found on the back surface 104 bof the film 104 in the step (c).

EXAMPLES

The present invention will be described in more detail with reference tothe following examples.

(Incubation of Pythium Torulosum)

Pythium torulosum, one of non-phytopathogenic pythiums, was inoculatedon a cornmeal agar culture medium together with dried turfgrass. Then,the culture medium was left at rest at a temperature of 25 degreesCelsius for 24 hours. Pythium torulosum was given by Professor Kageyama,who belongs to Gifu University River Basin Research Center. The driedturfgrass was provided by drying Korean lawn grass sterilized inaccordance with a high temperature and high pressure sterilizationmethod at 60 degrees Celsius for approximately 24 hours.

Then, the dried turfgrass to which a pseudohypha adhered was picked upfrom the culture medium. The thus-picked dried turfgrass was providedafloat to the pure water contained in a petri dish. The volume of thepure water was 20 milliliters.

After 18 hours, the water contained in the petri dish was observed usingan optical microscope. As a result, the present inventor confirmed thatzoospores of Pythium torulosum were released in the water contained inthe petri dish. In this way, an aqueous solution containing Pythiumtorulosum was provided. Hereinafter, this aqueous solution is referredto as “non-phytopathogenic aqueous solution”.

(Preparation of Culture Medium)

A potato dextrose agar culture medium melted at a high temperature wasadded to the second container 300. The potato dextrose agar culturemedium had a volume of 250 microliters. Then, the potato dextrose agarculture medium was turned into a gel at room temperature. In this way,the potato dextrose agar culture medium gel was provided as the solidculture medium 304.

A hydroponic culture solution (e.g., Otsuka-SA nutrient solution) havinga volume of 350 microliters was added as the liquid culture medium 302to the second container 300 containing the potato dextrose agar culturemedium gel. In this way, the second container 300 containing the liquidculture medium 302 and the solid culture medium 304 was prepared.

Inventive Example 1A

The first container 100 shown in FIG. 1 was prepared. This firstcontainer 100 was made of plastic. As shown in FIG. 2, the bottomsurface of the first container 100 was formed of a polyethyleneterephthalate film 104 (available from Merck KGaA, trade name: Millicell(registered trademark) PISP 12R 48). This polyethylene terephthalatefilm 104 comprised plural through-holes 104 c each having a diameter of3 micrometers. The plural through-holes 104 c were provided randomly inthe film 104. According to Merck KGaA, the diameter of the through-hole104 c may have a margin of error of approximately ± 10%.

Then, as shown in FIG. 6, the first container 100 was stacked on thesecond container 300. The back surface 104 b of the film 104 was incontact with the liquid culture medium 302. Subsequently, the hydroponicculture solution having a volume of 200 microliters was added to theinside of the first container 100. Furthermore, the non-phytopathogenicaqueous solution containing 200 zoospores of Pythium torulosum was addedto the inside of the first container 100.

The first container 100 was left at rest at a temperature of 25 degreesCelsius for 6 hours.

Subsequently, the first container 100 was separated from the secondcontainer 300. The non-phytopathogenic aqueous solution contained in thefirst container 100 was removed. Then, an agarose aqueous solutionhaving a concentration of 2% was added to the inside of the firstcontainer 100. The agarose aqueous solution was turned into a gel atroom temperature.

A fluorescent agent having oomycete combining ability (available fromBeckton Dickinson and Company, trade name: Calcofluor White (BD261195))having a volume of 600 milliliters was added to the inside of the secondcontainer 300. The final concentration of the fluorescent agent havingoomycete combining ability was 0.005%.

Then, the first container 100 was stacked on the second container 300again. The back surface 104 b of the film 104 was in contact with thefluorescent agent having oomycete combining ability. The first container100 was left at rest at 25 degrees Celsius for 10 minutes. Since the gelwas located in the first container 100, the fluorescent agent havingoomycete combining ability did not spread into the first container 100.

Subsequently, the first container 100 was separated from the secondcontainer 300. The fluorescent agent having oomycete combining abilitycontained in the second container 300 was removed. Then, a buffersolution was added to the inside of the second container 300. Thefollowing Table 1 shows components contained in this buffer solution andtheir concentrations.

TABLE 1 Component Concentration (mmol/L) NaCl 137 KCl 2.7 Na₂HPO₄ 10KH₂PO₄ 1.76

As shown in FIG. 9, the back surface 104 b of the film 104 was observedusing a fluorescent microscope 600 (available from Molecular DevicesJapan K.K. Trade name: ImageXpress MICRO). Table 2 shows filters and alens used for the fluorescent microscope 600.

TABLE 2 Excitation Band pass filter having a center wavelength of 377filter nanometers and a band width of 11 nanometers Fluorescence Bandpass filter having a center wavelength of 447 filter nanometers and aband width of 60 nanometers Object lens Magnification: 10times/Numerical aperture: 0.30

FIG. 10 is a microscope photograph of the back surface 104 b of the film104 in the inventive example 1A. As seen in FIG. 10, pseudohyphae ofPythium torulosum do not appear on the back surface 104 b. This meansthat the pseudohyphae of Pythium torulosum did not penetrate thethrough-holes 104 c.

Inventive Example 1B

In the inventive example 1B, an experiment similar to the inventiveexample 1A was conducted, except that each of the through-holes 104 chad a diameter of 1 micrometer. In particular, a polyethyleneterephthalate film 104 (available from Merck KGaA, trade name: MillicellPIRP 12R 48) was used.

FIG. 11 is a microscope photograph of the back surface 104 b of the film104 in the inventive example 1B. As seen in FIG. 11, pseudohyphae ofPythium torulosum do not appear on the back surface 104 b. This meansthat the pseudohyphae of Pythium torulosum did not penetrate thethrough-holes 104 c.

Comparative Example 1C

In the comparative example 1C, an experiment similar to the inventiveexample 1A was conducted, except that each of the through-holes 104 chad a diameter of 0.4 micrometers. In particular, a polyethyleneterephthalate film 104 (available from Merck KGaA, trade name: MillicellPIHT 12R 48) was used.

FIG. 12 is a microscope photograph of the back surface 104 b of the film104 in the comparative example 1C. As seen in FIG. 12, pseudohyphae ofPythium torulosum do not appear on the back surface 104 b. This meansthat the pseudohyphae of Pythium torulosum did not penetrate thethrough-holes 104 c.

Comparative Example 1D)

In the comparative example 1D, an experiment similar to the inventiveexample 1A was conducted, except that each of the through-holes 104 chad a diameter of 5 micrometers. In particular, a polyethyleneterephthalate film 104 (available from Merck KGaA, trade name: MillicellPIMP 12R 48) was used.

FIG. 13 is a microscope photograph of the back surface 104 b of the film104 in the comparative example 1D. As seen in FIG. 13, pseudohyphae ofPythium torulosum appear on the back surface 104 b. This means that thepseudohyphae of Pythium torulosum penetrated the through-hole 104 c.

Comparative Example 1E)

In the comparative example 1E, an experiment similar to the Inventiveexample 1A was conducted, except that each of the through-holes 104 chad a diameter of 8 micrometers. In particular, a polyethyleneterephthalate film 104 (available from Merck KGaA, trade name: MillicellPIEP 12R 48) was used.

Inventive Example 2

In the inventive examples 2A-2B and the comparative examples 2C-2E, anon-phytopathogenic aqueous solution containing zoospores of Pythiumcatenulatum was used in place of the non-phytopathogenic aqueoussolution containing zoospores of Pythium torulosum. Similarly to Pythiumtorulosum, Pythium catenulatum is also one kind of non-phytopathogenicpythiums. A non-phytopathogenic aqueous solution containing zoospores ofPythium catenulatum was prepared similarly to the case of thenon-phytopathogenic aqueous solution containing zoospores of Pythiumtorulosum.

Inventive Example 2A

In the inventive example 2A, an experiment similar to the inventiveexample 1A was conducted, except that the aqueous solution contained notPythium torulosum but Pythium catenulatum. Each of the through-holes 104c had a diameter of 3 micrometers.

Inventive Example 2B

In the inventive example 2B, an experiment similar to the inventiveexample 1B was conducted, except that the aqueous solution contained notPythium torulosum but Pythium catenulatum. Each of the through-holes 104c had a diameter of 1 micrometer.

Comparative Example 2C

In the comparative example 2C, an experiment similar to the comparativeexample 1C was conducted, except that the aqueous solution contained notPythium torulosum but Pythium catenulatum. Each of the through-holes 104c had a diameter of 0.4 micrometers.

Comparative Example 2D

In the comparative example 2D, an experiment similar to the comparativeexample 1D was conducted, except that the aqueous solution contained notPythium torulosum but Pythium catenulatum. Each of the through-holes 104c had a diameter of 5 micrometers.

Comparative Example 2E

In the comparative example 2E, an experiment similar to the comparativeexample 1E was conducted, except that the aqueous solution contained notPythium torulosum but Pythium catenulatum. Each of the through-holes 104c had a diameter of 8 micrometers.

Inventive Example 3

In the inventive examples 3A-3B and the comparative examples 3C-3E, anon-phytopathogenic aqueous solution containing zoospores of Pythiuminflatum was used in place of the non-phytopathogenic aqueous solutioncontaining zoospores of Pythium torulosum. Similarly to Pythiumtorulosum, Pythium inflatum is also one kind of non-phytopathogenicpythiums. A non-phytopathogenic aqueous solution containing zoospores ofPythium inflatum was prepared similarly to the case of thenon-phytopathogenic aqueous solution containing zoospores of Pythiumtorulosum.

Inventive Example 3A

In the inventive example 3A, an experiment similar to the inventiveexample 1A was conducted, except that the aqueous solution contained notPythium torulosum but Pythium inflatum. Each of the through-holes 104 chad a diameter of 3 micrometers.

Inventive Example 3B

In the inventive example 3B, an experiment similar to the inventiveexample 1B was conducted, except that the aqueous solution contained notPythium torulosum but Pythium inflatum. Each of the through-holes 104 chad a diameter of 1 micrometer.

Comparative Example 3C

In the comparative example 3C, an experiment similar to the comparativeexample 1C was conducted, except that the aqueous solution contained notPythium torulosum but Pythium inflatum. Each of the through-holes 104 chad a diameter of 0.4 micrometers.

Comparative Example 3D

In the comparative example 3D, an experiment similar to the comparativeexample 1D was conducted, except that the aqueous solution contained notPythium torulosum but Pythium inflatum. Each of the through-holes 104 chad a diameter of 5 micrometers.

Comparative Example 3E

In the comparative example 3E, an experiment similar to the comparativeexample 1E was conducted, except that the aqueous solution contained notPythium torulosum but Pythium inflatum. Each of the through-holes 104 chad a diameter of 8 micrometers.

Comparative Example 4

In the comparative examples 4A-4E, a phytopathogenic aqueous solutioncontaining zoospores of Pythium helicoides was used in place of thenon-phytopathogenic aqueous solution containing zoospores of Pythiumtorulosum. Unlike Pythium torulosum, Pythium helicoides is one kind ofphytopathogenic pythiums. A phytopathogenic aqueous solution containingzoospores of Pythium helicoides was prepared similarly to the case ofthe non-phytopathogenic aqueous solution containing zoospores of Pythiumtorulosum.

Comparative Example 4A

In the comparative example 4A, an experiment similar to the inventiveexample 1A was conducted, except that the aqueous solution contained notPythium torulosum but Pythium helicoides. Each of the through-holes 104c had a diameter of 3 micrometers.

Comparative Example 4B

In the comparative example 4B, an experiment similar to the inventiveexample 1B was conducted, except that the aqueous solution contained notPythium torulosum but Pythium helicoides. Each of the through-holes 104c had a diameter of 1 micrometer.

Comparative Example 4C

In the comparative example 4C, an experiment similar to the comparativeexample 1C was conducted, except that the aqueous solution contained notPythium torulosum but Pythium helicoides. Each of the through-holes 104c had a diameter of 0.4 micrometers.

Comparative Example 4D

In the comparative example 4D, an experiment similar to the comparativeexample 1D was conducted, except that the aqueous solution contained notPythium torulosum but Pythium helicoides. Each of the through-holes 104c had a diameter of 5 micrometers.

Comparative Example 4E

In the comparative example 4E, an experiment similar to the comparativeexample 1E was conducted, except that the aqueous solution contained notPythium torulosum but Pythium helicoides. Each of the through-holes 104c had a diameter of 8 micrometers.

Comparative Example 5

In the comparative examples 5A-5E, a phytopathogenic aqueous solutioncontaining zoospores of Pythium myliotaerum was used in place of thenon-phytopathogenic aqueous solution containing zoospores of Pythiumtorulosum. Unlike Pythium torulosum, Pythium myliotaerum is one kind ofphytopathogenic pythiums. A phytopathogenic aqueous solution containingzoospores of Pythium myliotaerum was prepared similarly to the case ofthe non-phytopathogenic aqueous solution containing zoospores of Pythiumtorulosum.

Comparative example 5A

In the comparative example 5A, an experiment similar to the inventiveexample 1A was conducted, except that the aqueous solution contained notPythium torulosum but Pythium myliotaerum. Each of the through-holes 104c had a diameter of 3 micrometers.

Comparative Example 5B

In the comparative example 5B, an experiment similar to the inventiveexample 1B was conducted, except that the aqueous solution contained notPythium torulosum but Pythium myliotaerum. Each of the through-holes 104c had a diameter of 1 micrometer.

Comparative Example 5C

In the comparative example 5C, an experiment similar to the comparativeexample 1C was conducted, except that the aqueous solution contained notPythium torulosum but Pythium myliotaerum. Each of the through-holes 104c had a diameter of 0.4 micrometers.

Comparative Example 5D

In the comparative example 5D, an experiment similar to the comparativeexample 1D was conducted, except that the aqueous solution contained notPythium torulosum but Pythium myliotaerum. Each of the through-holes 104c had a diameter of 5 micrometers.

Comparative Example 5E

In the comparative example 5E, an experiment similar to the comparativeexample 1E was conducted, except that the aqueous solution contained notPythium torulosum but Pythium myliotaerum. Each of the through-holes 104c had a diameter of 8 micrometers.

Comparative Example 6

In the comparative examples 6A-6E, a phytopathogenic aqueous solutioncontaining zoospores of Pythium aphanidermatum was used in place of thenon-phytopathogenic aqueous solution containing zoospores of Pythiumtorulosum. Unlike Pythium torulosum, Pythium aphanidermatum is one kindof phytopathogenic pythiums. A phytopathogenic aqueous solutioncontaining zoospores of Pythium aphanidermatum was prepared similarly tothe case of the non-phytopathogenic aqueous solution containingzoospores of Pythium torulosum.

Comparative example 6A

In the comparative example 6A, an experiment similar to the inventiveexample 1A was conducted, except that the aqueous solution contained notPythium torulosum but Pythium aphanidermatum. Each of the through-holes104 c had a diameter of 3 micrometers.

Comparative Example 6B

In the comparative example 6B, an experiment similar to the inventiveexample 1B was conducted, except that the aqueous solution contained notPythium torulosum but Pythium aphanidermatum. Each of the through-holes104 c had a diameter of 1 micrometer.

Comparative Example 6C

In the comparative example 6C, an experiment similar to the comparativeexample 1C was conducted, except that the aqueous solution contained notPythium torulosum but Pythium aphanidermatum. Each of the through-holes104 c had a diameter of 0.4 micrometers.

Comparative Example 6D

In the comparative example 6D, an experiment similar to the comparativeexample 1D was conducted, except that the aqueous solution contained notPythium torulosum but Pythium aphanidermatum. Each of the through-holes104 c had a diameter of 5 micrometers.

Comparative Example 6E

In the comparative example 6E, an experiment similar to the comparativeexample 1E was conducted, except that the aqueous solution contained notPythium torulosum but Pythium aphanidermatum. Each of the through-holes104 c had a diameter of 8 micrometers.

Comparative Example 7

In the comparative examples 7A-7E, an aqueous solution containing bothzoospores of Pythium torulosum and zoospores of Pythium helicoides wasused in place of the non-phytopathogenic aqueous solution containingzoospores of Pythium torulosum.

Comparative example 7A

In the comparative example 7A, an experiment similar to the inventiveexample 1A was conducted, except that the aqueous solution contained notonly Pythium torulosum but also Pythium helicoides. Each of thethrough-holes 104 c had a diameter of 3 micrometers.

Comparative Example 7B

In the comparative example 7B, an experiment similar to the inventiveexample 1B was conducted, except that the aqueous solution contained notonly Pythium torulosum but also Pythium helicoides. Each of thethrough-holes 104 c had a diameter of 1 micrometer.

Comparative Example 7C

In the comparative example 7C, an experiment similar to the comparativeexample 1C was conducted, except that the aqueous solution contained notonly Pythium torulosum but also Pythium helicoides. Each of thethrough-holes 104 c had a diameter of 0.4 micrometers.

Comparative Example 7D

In the comparative example 7D, an experiment similar to the comparativeexample 1D was conducted, except that the aqueous solution contained notonly Pythium torulosum but also Pythium helicoides. Each of thethrough-holes 104 c had a diameter of 5 micrometers.

Comparative Example 7E

In the comparative example 7E, an experiment similar to the comparativeexample 1E was conducted, except that the aqueous solution contained notonly Pythium torulosum but also Pythium helicoides. Each of thethrough-holes 104 c had a diameter of 8 micrometers.

The following Table 3-Table 9 show the number of the pseudohyphae whichpenetrated the through-hole 104 c in the inventive example 1A—thecomparative example 7E.

TABLE 3 Number of the pseudohyphae which Diameter of penetrated theContained through-hole through-hole Pythium (micrometer) 104c Inventiveexample 1A Pythium 3.0 0.0 Inventive example 1B torulosum 1.0 0.0Comparative example 1C 0.4 0.0 Comparative example 1D 5.0 7.0Comparative example 1E 8.0 6.0

TABLE 4 Number of the pseudohyphae which Diameter of penetrated theContained through-hole through-hole Pythium (micrometer) 104c Inventiveexample 2A Pythium 3.0 0.0 Inventive example 2B catenulatum 1.0 0.0Comparative example 2C 0.4 0.0 Comparative example 2D 5.0 11.3Comparative example 2E 8.0 5.5

TABLE 5 Number of the pseudohyphae which Diameter of penetrated theContained through-hole through-hole Pythium (micrometer) 104c Inventiveexample 3A Pythium 3.0 0.0 Inventive example 3B inflatum 1.0 0.0Comparative example 3C 0.4 0.0 Comparative example 3D 5.0 7.5Comparative example 3E 8.0 23.5

TABLE 6 Number of the pseudohyphae which Diameter of penetrated theContained through-hole through-hole Pythium (micrometer) 104cComparative example 4A Pythium 3.0 18.0 Comparative example 4Bhelicoides 1.0 11.0 Comparative example 4C 0.4 0.0 Comparative example4D 5.0 94.7 Comparative example 4E 8.0 59.7

TABLE 7 Number of the pseudohyphae which Diameter of penetrated theContained through-hole through-hole Pythium (micrometer) 104cComparative example 5A Pythium 3.0 48.7 Comparative example 5Bmyliotaerum 1.0 1.5 Comparative example 5C 0.4 0.0 Comparative example5D 5.0 84.0 Comparative example 5E 8.0 31.3

TABLE 8 Number of the pseudohyphae which Diameter of penetrated theContained through-hole through-hole Pythium (micrometer) 104cComparative example Pythium 3.0 21.6 6A aphanidermatum Comparativeexample 1.0 11.6 6B Comparative example 0.4 0.0 6C Comparative example5.0 53.8 6D Comparative example 8.0 17.6 6E

TABLE 9 Number of the pseudohyphae which Diameter of penetrated theContained through-hole through-hole Pythium (micrometer) 104cComparative example 7A Pythium 3.0 23.0 Comparative example 7B torulosum1.0 16.0 Comparative example 7C and 0.4 0.0 Comparative example 7DPythium 5.0 90.3 Comparative example 7E helicoides 8.0 42.0

As is clear from Table 3-Table 9, when the through-hole 104 c has adiameter of not less than 1 micrometer and not more than 3 micrometers,the phytopathogenic pythium appears on the back surface 104 b of thefilm 104. On the other hand, within this range of diameter,non-phytopathogenic pythium does not appear on the back surface 104 b ofthe film 104. Therefore, the phytopathogenic pythium 202 appears on theback surface 104 b selectively in a case where the through-hole 104 chas a diameter of not less than 1 micrometer and not more than 3micrometers. In other words, the phytopathogenic pythium 202 appearsoutside of the container 100 selectively.

As is clear from Table 3-Table 9, when the through-hole 104 c has adiameter of 0.4 micrometers, neither the non-phytopathogenic pythium northe phytopathogenic pythium appears on the back surface 104 b of thefilm 104.

As is clear from Table 3-Table 9, when the through-hole 104 c has adiameter of not less than 5 micrometers, not only the phytopathogenicpythium but also the non-phytopathogenic pythium appears on the backsurface 104 b of the film 104.

Therefore, if the through-hole 104 c has a diameter of not less than 1micrometer and not more than 3 micrometers, the pseudohyphae of thenon-phytopathogenic pythium does not appear on the back surface 104 b ofthe film 104, whereas the pseudohyphae of the phytopathogenic pythiumappears on the back surface 104 b of the film 104. Using thisdifference, a skilled person can determine whether or not all of thepythiums contained in the test sample are non-phytopathogenic pythium.

INDUSTRIAL APPLICABILITY

The present invention can be used to determine easily whether or not allof the pythiums contained in a test sample such as agricultural water orsoil are non-phytopathogenic.

REFERENTIAL SIGNS LIST

-   100 First container    -   102 Flange        -   104 Film            -   104 a Front surface            -   104 b Back surface            -   104 c Through-hole-   200 Test sample-   202 Phytopathogenic pythium    -   202 a Part of Phytopathogenic pythium-   300 Second container    -   302 Liquid culture medium    -   304 Solid culture medium-   402 fluorescent agent having oomycete combining ability-   500 Light source-   600 Microscope

The invention claimed is:
 1. A method for determining whether or not allpythiums contained in a test sample are non-phytopathogenic, the methodcomprising: (a) putting the test sample on a front surface of a filmcomprising a through-hole having a cross-sectional area of not less than0.785 square micrometers and not more than 7.065 square micrometers; (b)leaving the test sample at rest for six hours after the step (a); (c)detecting the presence of pseudohyphae on a back surface of the filmopposite to the front surface after the step (b); and (d) determiningthat all the pythiums contained in the test sample arenon-phytopathogenic, as a result of a detection that pseudohyphae is notfound on the back surface of the film in the step (c).
 2. The methodaccording to claim 1, wherein the pythiums contained in the test sampleis at least one selected from the group consisting of Pythium torulosum,Pythium catenulatum, and Pythium inflatum.
 3. The method according toclaim 1, further comprising: a step of bringing the back surface of thefilm into contact with a fluorescent agent for dyeing the pythiumsbetween the step (b) and the step (c).
 4. The method according to claim3, further comprising: a step of turning the test sample into a gelbefore the back surface of the film is brought into contact with thefluorescent agent for dyeing the pythiums.
 5. The method according toclaim 1, further comprising: a step of supplying a culture medium to thetest sample prior to the step (b).
 6. The method according to claim 5,wherein the culture medium is a liquid culture medium.
 7. The methodaccording to claim 5, wherein the culture medium is a solid culturemedium.
 8. The method according to claim 1, wherein the test sample isleft at rest while the back surface of the film is in contact with theculture medium in the step (b).
 9. The method according to claim 1,wherein the film has a thickness of not less than 10 micrometers and notmore than 100 micrometers.
 10. The method according to claim 1, whereinthe film comprises a plurality of the through-holes.
 11. The methodaccording to claim 1, wherein the test sample is solid.
 12. The methodaccording to claim 11, wherein the solid test sample is at least oneselected from the group consisting of soil and a crushed plant.
 13. Themethod according to claim 1, wherein the test sample is liquid.
 14. Themethod according to claim 13, wherein the liquid test sample is at leastone selected from the group consisting of agricultural water, a liquidused for hydroponic culture, a liquid used for washing a plant, a liquidextracted from a plant, a liquid used for washing an agriculturalmaterial, and a liquid used for washing clothing or a shoe.
 15. Themethod according to claim 1, further comprising: (e) determining that atleast one of the pythiums contained in the test sample isphytopathogenic, as a result of the detection that pseudohyphae is foundon the back surface of the film in the step (c).
 16. The methodaccording to claim 1, wherein the (c) comprises, observing the backsurface by a microscope and irradiating the front surface with light.17. The method according to claim 1, wherein the film is an organicresin.
 18. The method according to claim 17, wherein the organic resinis polyethylene terephthalate.
 19. The method according to claim 1,wherein the through-hole has a shape of a cylinder.